Aug., 1951
NATURAL PROTEOSES ISOLATED FROM OILSEEDS AND NUTS
Poly-DL-arginine F1avianate.-A
solution of flavianic acid
(5 g.) in water (5 ml.) was added to poly-Dtarginine sulfate (1 9.) in water (2 ml.) and the mixture left in the refrigerator for 48 hours. The supernatant liquid was decanted and the residue washed with cold water and alcohol and dried in vacuo over sulfuric acid. It was dissolved in hot dimethyl formamide (2 d.), the solution iiltered and poured into absolute ethanol (75 ml.). The yellow flocculent precipitate of poly-m-arginine flavianate which separated out was centrifuged, washed several times with absolute ethanol and cther and dried in vacuo over sulfuric acid; yield quantitative, m.p. 214-220' (dec.). Anal, Calcd. for poly-DL-arginine flavianate (n average = 80): C, 40.8; H, 3.8; N, 17.9; amino N, 0.04; S, 6.8. Found: C, 40.3; H, 4.1; X, 17.9; amino N, 0.5; S, 7.2.
3995
Total Hydrolysis of Poly-m-arginine F1avianate.-PolyDL-arginine flavianate was hydrolyzed with 10% hydrochloric acid at 120' for 6 hours and the arginine content of the neutralized hydrolysate determined enzymatically and microbiologically. Anal. Calcd. for 100 mg. poly-m-arginine flavianate ( n = 80): targinine, 18.5 mg. Found: c a r g i n h e , 19.6 mg. (enzymatic method)," 18.0 mg. (microbiological method) . I J
Acknowledgment.-The authors are indebted to Miss S. Sicher for carrying out the microbiological assays. REHOVOT, ISRAEL
RECEIVED JANUARY 25, 1951
[CONTRIBUTION FROM ALLERGEN RESEARCHDIVISION,BUREAUOF AGRICULTURAL A N D INDUSTRIAL CHEMISTRY, U. S. DEPARTMENT OF AGRICULTURE]
The Chemistry of Allergens. XI. Properties and Composition of Natural Proteoses Isolated from Oilseeds and Nuts by the CS-1A BY JOSEPH R. SPIES,E. J. COULSON, DORRISC. CHAMBERS, HARRYS. BERNTON, HENRYSTEVENS AND JAMES H. SHIMP The procedure used to isolate the principal allergen of cottonseed, CS-lA, has been applied to the following oilseeds and nuts: Almonds, Brazil nuts, castor beans, coconuts, Barcelona and DuChilly filbert nuts, flaxseed, kapok seed, black mustard seed, peanuts, pecan nuts, soybeans, tung nuts and black walnuts and English walnuts. Typical natural proteoses possessing similar chemical, allergenicand antigenic properties were obtained from almond nuts, Brazil nuts, castor beans, cottonseed, Barcelona and DuChilly filbert nuts, flaxseed, kapok seed and mustard seed. Soybeans and peanuts yielded immunologically atypical fractions, and black walnuts yielded a fraction having immunological properties intermediate between those of the typical and atypical groups. The chemical composition, allergenic and antigenic properties of these substances are described. It is suggested that these substances be recognized as a class of native proteins and that they be designated by the term natural proteose proposed by Wells and Osborne, or the term natural proteone to show that, while these substances have chemical DroDerties similar to those of non-antigenic derived proteoses or peptones they are highly antigenic preformed components of-the seeds.
It has long been known that multiple sensitivity to cottonseed, nuts and other oilseeds is a frequently encountered clinical grouping. a Consideration of this clinical classification, after isolation of the principal allergen of cottonseed, CS-14 or CS-1A,6 led to the assumption that allergens of other oilseeds and nuts might be similar enough to be isolated by a general pro~edure.~JThe validity of this speculation was shown by the isolation of the ricinfree allergenic fraction CB-1A from castor beans' by the same procedure originally developed for the isolation of CS-1A from botanically unrelated cottonseed. CB-1A was remarkably similar in composition and chemical and immunological properties to CS-lA, although differing in allergenic specificity. Kapok seed, which is botanically related to cottonseed, also yielded a similar allergen, KS-lA.* CS-1A and CB-lA, although not homogeneous, are important because they contain the most potent allergens of respective seeds, completely freed from other allergens and antigens present in the seeds. This has been conclusively shown by extensive (1) Not subject to copyright.
(2) Paper X, Spies, Coulson and Stevens, THIS JOURNAL, 66, 1798 (1944). (3) Bowman and Walzer, "Asthma and Hay Fever in Theory and Practice," Charles C. Thomas, Baltimore, Maryland, 1931, p. 394. (4) Spies, Bernton and Stevens, J . Allcrgy, 10, 113 (1939). (5) Spies, Coulson, Bernton and Stevens, THXS JOURNAL, 62, 1420 (1940) 03) Spies, ibid., 63,2994 (1941). (7) Spies and Coulson, i b i d . , 66, 1720 (1943). (8) Coulson, Spies and Stevens, J. Iinmunol , 49,99 (1944). KS-1A contained a minor component that was serologically identical with CS-1A from cottonseed.
passive transfer and cutaneous tests on sensitive human subject^^^^^ and by gross anaphylaxis and Schultz-Dale tests with guinea pigs."-14 CS-1A and CB-1A provided relatively abundant and highly concentrated source materials for further studies designed to obtain homogeneous allergens or allergens sufficiently purified to permit determination of the chemical nature of the active components with reasonable certainty. The essentially carbohydrate-free natural proteoses15 CS6OCI6 and CB-65A,17 possessing full allergenic activity, were isolated from CS-1A and CB-1A and all evidence adduced showed that the allergenic and immunological specificities of CS-1A and CB-1A were inherent in the type of substance represented by CS-6OC and CB-65A, respectively. To determine the distribution of allergenic natural proteoses, several more oilseeds and nuts were subjected to the CS-1A procedure. The present paper describes results of this study with almond nuts, Brazil nuts, castor beans, cottonseed, coconuts, filbert nuts, flaxseed, kapok seed, mustard seed, pecan nuts, peanuts, soybeans, tung nuts, black walnuts and English walnuts. (9) Bernton, Spies and Stevens, J . Allergy, 13,289 (1942). (10) Spies, Chambers, Bernton and Stevens, ibid., 14, 7 (1942). (11) Coulson, Spies and Stevens, J . Zmmunol., 41, 375 (1941). (12) Coulson, Spies and Stevens, ibid., 16, 347 (1943). (13) Coulson and Spies, ibid., 46, 367 (1948). (14) Coulson, Spies and Stevens, J . Alkrgy. 41, 34 (1950). (16) Term proposed by Wells and Osborne, 1.Znfectious Diseases, 17, 239 (1916). (16) Spies and Umberger, THIS JOURNAL, 64, 1889 (1942). (17) Spies, Coulson,Chambers, Bernton and Stevens, ibid., 66, 748 (1944).
VOl. 73
SPIES,COULSON, CHAMBERS, BERNTON, STEVENS AND SHIMP 'rAi3LE
1
SUBSTANCES EXAMINED FOR ALLERGENS, &,lET€IOD U P ISOLATXOS, YIELD AND ANALYSES Method Solvent used for defatting
Source
Substance
Almond nuts-Prunus amygdalus Batsch. Brazil nuts-BerthoEletia excelsa Humb. & Bonpl. Castor beans-Ricinus communis 1,. Castor beans-Ricinus communis L. Cottonseed-Gossypiuin spp. Coconuts-Cocos nuczfera L. Filbert nuts-(Barcelona)-Corylzis avellana L. Filbert nuts-( Barcelona)-Corylus avellana L. Filbert nuts-( DuChi1ly)-Corylus avellana I,. Flaxseed-Linum usifatissimum L.
isolaof Yield aller-of NitroAnalyses Chrbo% ' gen hydrate* tion geo," %
Commercial Commercial
Pet. ether Pet. ether
1A 1A
0.46 0.81
16.9 17.6
12.6 6.9
U. S. Dept. Agr. Commercial (Brazilian Pomace) So. Reg. Res. Lab.
Ethyl ether S o t extracted
1A 1C
1.76 0.33
18.4 17.1
3.1 8.0
1A
1.38
12.1
36.4
1A 1A
..
U.S. Dept. Agr.
Depigmented and defatted Ethyl ether Ethyl ether
0.006 .30 17.1
.. 10.6
Coinmercial
Pet. ether
H1B
.26
18.8
6.6
Commercial
Pet. ether
H1B
.33
19.2
6.0
Commercial
Dehulled and defatted Benzene Ethyl ether Ethyl ether Ethyl ether Ethyl ether
1A
1.98
11.3
39.4
Commercial
34.5 1A 0.78 12.6 Commercial 40.7 1A 0.58 11.1 Commercial IA .07 15.4 16.7 Commercial 1B .18 15.2 10.6 Commercial 1A .00 .. .. U. S. Pecan Field Lab. Albany, Georgia 20.4 1B .10 13.3 U. S. Regional Soybean Ethyl ether Lab., Urbana, Illinois .. Xthyl ether 1A .008 5 . 7 Commercial Tung nuts-Aleurites fordii Hemsl. 11.2 Ethyl ether 1A .40 17.9 Commercial Walnuts (Black) Jugluns nigra (L.) .. Ethyl ether 1A .OO Commercial IValnuts (English) Juglans regia (L.) Calculated as per cent. of ash-free and water-free allergen in air-dried defatted meal. * Determined by the method of M. Sorensen and Haugaard as modified by Heidelberger and Kendall, J . Immunol., 30 267 (1936). Galactose [ C X ] ~ D$79.7 This sample was contaminated with 3 t o 4y0B. arvensis. c, 3.0 g. 100 ml.) was used as standard.
Kapok-Ceiba pentandra (L.) Gaertn. Mustard seed"-Brassica nigra (L.) Koch Peanuts-A rachis hypogaea L. Peanuts--A rachis hypogaea L. Pecan nuts-Carya illinoensis (Wang.) Koch. Soybeans--Glycine inax (I,,) Merrill
Experimental Source of Materials and Preliminary Treatment.-The 5eeds and nuts used in this study were obtained from commercial sources and government experiment stations, as shown in Table I. Identification and scientific names were obtained through the cooperation of S. F. Blake, H. I,. Crane, F. J. Herman and Miss A. F. Musil of the Bureau of Plant Industry, Soils and Agricultural Engineering, U. S. Department of Agriculture. Seeds and nuts were ground and then defatted by thorough percolation a t room temperature with the solvents shown in Table I. Cottonseed that had been defatted and depigmented at the Southern Regional Research Laboratory by a special process1*was a better source of C S l A than was cottonseed that had been simply defatted with either benzene or ether, because less difficulty with stable suspensions was encountered in the isolation procedures. Commercial castor bean pomace has the advantage that preliminary grinding and defatting of toxic castor beans can be avoided, but the disadvantage that allergen-destructive treatment may have occurred before the pomace mas received. CB-1A isolated from one lot of Brazilian pomace had the same nntigenic activity as that obtained from untreated domestic castor beans,z but that obtained from :i later lot of pomace had a lower antigenic potency. Flaxseed was difficult to handle because of the mucilaginous constituents in the hulls, and a relatively low yield of flaxseed-lA was obtained from the first lot studied. The flaxsted-lA described in this paper was obtained from seed dehulled and defatted by a flotation process t o be described elsewhere. No difficulty was caused by mucilaginous components when dehulled seeds were used. Isolation of Allergens by the CS-IA Procedure .-Fundaitlentally the same procedure was used to isolate the allergetis f t oin the seeds and nuts reported herein as wrts used to isoLite ___'IS) I I o r t n e , a n d I-i ,I' O i l
/I, ,/
sorip 23, 12: iI'111,
C S l A from cottonseed4s6and CB-1A from castor beans.' This procedure was based on the discoveries that the cottonseed allergen is soluble in water, soluble in 25% ethanol but insoluble in 75% ethanol, stable in boiling water and nonprecipitable by basic lead acetate. Any modifications in procedures used were made with consideration of the abovementioned properties of the allergens. The previously published CS-1A procedure is not described here, but modifications and coinrneiits pertinent to its application are given below. Weighed quantities of defatted meals were stirred mechanically for three hours or longer with distilled water in the proportions of 6 to 10 liters of water to 1 kilogram of meal for the initial extraction. Some extracts were concentrated by evaporation in a current of air instead of by vacuum distillation. Chloroform was used as preservative during such evaporations. Precipitation with excess basic lead acetate is a critically important step in obtaining allergens immunologically free from other allergens and antigens in the seeds and nuts. I t is essential that excess lead be used for the precipitation of these other allergens and that the lead salts be completely separated. To determine the quantity of basic lead acetate required for complete precipitation, 20 ml. of the appropriate solution was placed in a 40-ml. centrifuge tube, and 3 ml. of basic lead acetate solution was added with stirring. The suspension was centrifuged, and another portion of basic lead acetate was added to the clarified supernatant solution. The point where no precipitate formed was determined by repeated tests and the calculated amount of basic lead acetate was then added t o the main solution. In the previously described procedure, 10% basic lead acetate (the type used for sugar analysis) in 25% ethanol solution was used. The precipitation may also be carried out in water solution by using 10% basic lead acetate in water. The bulk of precipitated lead salts was removed by centrifugiiig. If colloidal lead salts remained in solution after Illis preliininary centrifuging, it was found convenient, In
Aug., 1951
NATURAL PROTEBES ISOLATED
most cases, to remove them by Seitz filtration (Size 14 filter). However, some extracts were too viscous for Seitz filtration and could only be clarified by supercentrifuging. Excess lead was removed from clarified solutions by adding 10% sodium carbonate until the PH of the solution was 9.4 to 9.8. Precipitated lead carbonate was removed by centrifuging, and any colloidal lead carbonate was removed by Seitz filtration or by centrifugation in the batch bowl of the Sharples supercentrifuge, care being used to avoid undue heating. Clear solutions were tested for complete removal of lead by further addition of a few drops of sodium carbonate solution. The pH of solutions was adjusted to about 6.2 with 50% acetic acid solution as soon as possible after removal of lead, brom cresol purple being used as indicator. Preparations obtained in this way were designated by the suffix -lA. “1B” Procedure.-In some cases, a niilder isolation procedure was used by avoiding heating and adjusting the solution to PH 9.6. In these cases, suspended protein in the original water extract was flocculated by bringing the PH to about 4.7 with 50% acetic acid solution. The precipitate was removed by centrifuging and discarded. The pH of the solution was then adjusted to about 5.6 with sodium hydroxide solution either before or after concentration by evaporation a t room temperature. Excess lead in these cases was removed by hydrogen sulfide precipitation. The rest of the procedure was the same as the 1A procedure. These preparations were designated by the suffix -lB. Heated 1B Preparations.-Samples of Barcelona and DuChilly filbert nut-1B were dissolved in water (about 1% solution) and heated in a boiling water-bath for 1 hour. The solutions were cooled, and the small amount of coagulum was centrifuged off and discarded. The solutions were filtered and made 0.1 N in sodium acetate buffered a t PH 5.6. The allergens were precipitated with three volumes of ethanol and isolated as usual. Recoveries of 81 and 85% were made from Barcelona filbert-1B and DuChilly filbertl B , respectively. The heat treatment had no evident effect on immunologic potency or specificity. These preparations were designated by the suffix -HlB. “1C” Procedure.-In this procedure heat was used as in the 1A procedure but hydrogen sulfide, instead of sodium carbonate, was used to precipitate excess lead. Considerable difficulty was sometimes experienced in removing the colloidal lead sulfide in this method. There was no indication that the use of sodium carbonate caused any decrease in potency of the allergens. These preparations were designated by the suffix -lC. All allergen fractions were finally dried in a vacuum desiccator over phosphorus pentoxide or calcium chloride: the latter was preferred because slight discoloration occurred on the surface of some samples dried with phosphorus pentoxide. Samples were ground to pass 60, 80 or 100 mesh sieves and equilibrated with air before analysis. All of the fractions were practically white, and all, except mustard-la, were soluble in water. Mustard-1A contained some denatured material and was only partially soluble. Determination of Amino Acids.-Amino acids were determined microbiologically, except that tryptophan was determined by general procedure N as described by Spies and Chambers. le Microorganisms were obtained from the American Type Culture Collection, 2029 M St., Washington, D. C. Hydrolysis.-One hundred millikams of sample was dissolved in 2 ml. of 3 N hydrochloric acid (prepared from constant boiling acid) in a 17 X 90 mm. Pyrex tube. After complete solution a t room temperature, the tubes were placed upright in a Pyrex desiccator t o which had been added enough water to fill the desiccator with water vapor. The desiccator was evacuated t o 40 mm. pressure and heated in an autoclave for 8 hours at 15 pounds pressure (120-125’). The hydrolysate was cooled and filtered quantitatively through a hardened paper, water being used for washing and making the final volume 20 ml. Suitable dilutions of this solution were used for analyses. Amino acids in the hydrolysates were considered to be in the L-form. Amino Acid Standards.-Best quality commercial, specially prepared or recrystallized amino acids were used for standards. Each amino acid so used had the theoretical nitrogen content, as determined by the Kjeldahl micromethod. Gforms of arginine, recrystallized cystine, glu(19) Spies and Chambers, Anal. Chcm., 31, 1249 (1949).
FROM
OILSEEDSAND NUTS
3997
tamic acid, histidine, proline and tyrosine were used. LMethionine was prepared as previously described.MBz1 Samples of L-serine, L-threonine and &-valine, resolved by enzymatic hydrolysis of respective chloroacetyl derivative,aZ were obtained through the kindness of Dr. J. P. Greenstein. DGAlanine and glycine were recrystallized. Except for aspartic acid only the L-forms in standard DL-mixtures were utilized by the microorganisms. Under the conditions of the test both forms of aspartic acid were utilized. Method of Analysis.-Essentially the method and medium of Henderson and &ellz3 were used. The PH of the sample was adjusted to that of the medium before mixing. The volume of the medium was 1 ml., aiid that of sample was also 1 ml. Organisms were allowed to grow for 66 to 7 2 hours in the dark at 30’. The samples were then diluted to 50 ml., and the lactic acid was titrated with 0.05 N sodium hydroxide, brom thymol blue being used as indicator. A t least two determinations were made a t each of five concentration levels. Conventional standard curves were obtained. Results were calculated from at least three concentration levels that fell in the useful part of the standard curve. The determinations of alanine and cystine were not as satisfactory as the others by the method used although good results were obtained. Evaluation of Reliability of Results .-Microbiological determination of amino acids has not yet become standardized. Therefore, evaluation of the accuracy of the results was made by comparison with values obtained by other workers using a different method. This was done by determination of the amino acids in crystalline B-lactoglobulin2‘ and comparing results with those of Stein and Moorez6 who used partition chromatography on starch columns (Table 111). The eighteen amino acids determined accounted for 96.4% of the total nitrogen, exclusive of amide or ammonia nitrogen. When the value of amide nitrogen used by Stein and Moore was added the total recovery of nitrogen was 103.3% as compared to 99.6% obtained by Stein and Moore. ‘ Individual percentage differences between values obtained in this work and those of Stein and Moore ranged from 0 to 35.4% while the average deviation was =!=10.9%. The amino acid content of casein26 was also determined, and exclusive of amide nitrogen 96.5% of the total nitrogen was accounted for in the eighteen amino acids determined. In general, good agreement of values with those recently published by Gordon, el a1.,27and Heudersou and SnellZawas obtained. Further evidence of the reliability of the microbiological values for arginine and tyrosine was furnished by comparison with the values obtained chemically on three representative allergen preparations. Arginine was isolated as the analytically-pure monoflavianate (shown by nitrogen and sulfur analysis) by the method of Vickery.*S Tyrosine was determined colorimetrically by the method of Lugg.20 The values obtained by microbiological and chemical methods are in good agreement as shown in Table IV. In view of the foregoing considerations, the values obtained microbiologically for the amino acids are regarded as entirely satisfactory for demonstrating the composition of the allergen preparations. Other Analytical Methods.-Nitrogen was determined by a Kjeldahl micromethod using mercuric sulfate as catalyst. Arginine monoflavianates were reduced with hydriodic acid before Kjeldahl digestion. Sulfur was determined by the micromethod of Pregl. Water was determined by heating the sample for 3 hours in an Abderhalden vacuum drier at 110’ followed by weighing in a closed system. Ash was determined by ignition in an electric furnace for l .5 hours, during which time the temperature rose to 750’. All analytical results are expressed on an ash-free and water-free basis. Cutaneous Tests.-A scratch that just penetrated the epidermis was made on the forearm or thigh of the sensitive (20) Spies, J . B i d . Chem., 184, 439 (1950). (21) Spies and Chambers, ibid.. 188, 709 (1950). (22) Price, Gilbert and Greenstein, ;bid., 179, 1169 (1949). (23) Henderson and Snell, ibid., 173, 15 (1948). (24) The sample used was described in reference 19. (25) Stein and Moore, J . Biol. Chcm., 178, 79 (1949). (26) Prepared a s described in reference 19. (27) Gordon, Semmett, Cable and Morris, T H i s JOURNAL., 71, 3293 (1949). (28) Vickery, J. B i d . Chew., 183, 325 (1840). (29) Lugg, Biochcm. J . , 81, 1422 (1937); ;bid., 84, 778 (1938).
Vol. 73
3995 TABLE I1 AMINOACIDCONTENT OF OILSEEDALLERGEKS Results expressed as per cent. of total nitrogen of the allergen in the indicated amino acid AsSubstance .Umond-1 A Brazil nut-1A Castor bean-1.t (Brazilian) Castor bean-lA !domesticIe Cottonseed-1-4 Filbert nut-1.4 (Barcelona) Filbert nut-H1B (Barcelona) l'ilbert nut-IIlIi (DuChilly) Flaxseed- 1 A Kapok-1A Mustard-lA(Black) Peanut-1B Soybean-li\ Walnut-1A (Black)
partic nine5 acid0 i . 9 4.0 .';.b 3.7
Ala-
Argi-
Glu- Histamic tiacidd dine' 19.2 4 . 2
Iso-
Me-
Phenyl-
'rrypto- Tyro- Valcineacinea sinea nines ninea line' inea oninea phan sinea ine' Total 0.8 1 . 2 87.3 1.0 1 4 7 . 5 1 . 1 0 . 7 1 . 4 2 . 6 2 . 4 0.3 0.7 0 . 5 90.2 16fi 4.2 0.4 2 7 2.3 5 2 .:i3 . 4 2 . 9 0 . 0 0 . 0
leu- Leu- Ly- thio- ala- Pro- ser- T h e -
35.1
Glytinee cine" 3.7 12.3 2.7 3.6
?!a,!>
2.1
i.8
1.3 :?.I
,*I,!!
21 1
:!O
205
3 4
1.6 2.7 2 , s 0 5 1.0
-%.$a !K>
?:Ll
O!J
ti
? t i
1,;
2.4
0.2
?it ii
1 ;-;
ii
2 0
::(I
1 li
.I
nineb 18.4
o!i
;I8
i i
7 1
2 . 1 2i.i 5 . 2 27..-1
Y I
4 1
Cys-
:IS(, 3.tt
6 . 5 -L I
:i(i!t
:j,l
,;.!I
4.0
:3!1.4
:1.0
8.5
5.(i
X . 2
1.7
8.7 6.8 11.7 6.4
3.6 5.0 8.7 3.4
17.7 10.8 17.6 36.8
-cw,
lJ'
:
